New antituberculotic compounds and process of preparing same



.straius resistant against 274-6 0. (dec.).

United States Patent 1 2,758,117 NEW ANTITUBE'RCULOTIC COMPOUNDS AND PROCESS OF PREPARING SAME Arthur Ernest Wilder Smith, Wolhusen, Lucerne, Switzerland, assignor to Ed Geistlich Sohne A. G. fur

Chemische Industr'ie, Wolhusen, Luceme, Switzerland No Drawing. Application November '15, 1954, 'Serial No. 469,056 4 Claims. (Cl. 260-295) This invention is concerned with improvements in or relating to new anti-tuberculotic compounds 'efiective in the treatment of human and animal tuberculosis.

In recent years a great deal of research has been carried out to discover chemotherapeutic agents having inhibiting action against the micro-organisms responsible for tuberculosis in humans and animals, i. e. M. tuberculosis. This research has revealed a number of compounds (called for convenience tuberculostatics) which have found varying degrees of favour, some of the more important being streptomycin, p-amino salicylic acid and isonicotinic acid hydrazide and derivatives thereof.

Various drawbacks have however been found to be attendant upon the use of the tuberculostatics hitherto proposed for the treatment of tuberculosis. Thus, for example streptomycin and p-amino salicylic acid often give rise to undesirable side effects; most of such tuberculostatics furthermore, including iso-nicotinic acid hydrazide, suifer, in varying degrees, from the serious defeet that the tubercle bacillus develops a resistance against the tuberculostatic. This development of resistance may take place quite quickly after the commencement of therapy and may often produce a condition which is more diflicult to treat than the original condition. Attempts have been made to overcome this last mentioned defect by the use of two or more different tuberculostatic compounds either together or consecutively, but with this procedure, the treatment requires to be carefully controlled and frequent observations of the patient must be made.

It may be said therefore that an ideal tuberculostatic should have, inter alia, the following characteristics:

1. It should have a low toxicity and should give rise to substantially no side efiects such as nausea, vomiting, dizziness, etc.

2. It should have high inhibiting action in vivo against the tubercle bacillus.

3. It should not easily induce the bacillus to form it, and should be active against strains of tubercle bacillus already resistant against known tuberculostatics.

4. It should be stable and capable of ready administration preferably per 0s. 7,

We have now found certain new compounds which more nearly approach the requirements of an ideal tuberculostatic than compounds hitherto used for this purpose.

The parent substance of our new compounds has the empirical formula CIHN302 and may be obtained by the reaction of isonicotinic acid hydrazide with phos'gene. -We believe the compound to be Z-pyridyl- (4) -l 3 :4=osydiazolone-(5) which may be represented by the struc- 'tural formula bases which salts and salt-like compounds have in general ,the tuberculostatic activity of the parent substance.

Our new parent compound (which for convenience we shall refer to as the diazolone) has a melting point of tain, and is of course liable to variation with purity.

This melting point was determined 'for the purest material we have so far been able to oh- 2,758,117 Patented Aug. 7, 1956 As stated above, the diazolone is capable of forming salts or salt like compounds with acids and bases. Thus on the one hand acid addition salts may be formed from for example hydrochloric, sulphuric, tartaric and citric acids. Wit-h bases, such as metal hydroxides (or 'by metathesis with metal salts) base addition salts may be formed. Of particular interest are the compounds of copper, nickel, iron, cobalt manganese and .gold. We use the term salt or salt-like compounds to designate the compounds which are obtained by salt-forming procedures applied to the diazolone; such compounds may not in fact be true salts but rather complex or addition-compounds the nature of which is not entirely clear.

The salts or salt-like compounds with which this invention is concerned are the non-toxic salts i. e. such salts (or salt-like compounds) as are substantially nontoxic at therapeutic dosage although it will be understood that the radical used to form a salt or salt-like derivative with the diazolone may itself contribute to the toxicity of such derivative.

We have further found one particular derivative of the diazolone to be particularly advantageous in the treatment of tuberculosis namely, the compound which may beobtained by reacting our new compound with p-amino salicylic acid (or by metathetically reacting a salt of our new compounds with a p-amino salicylic acid salt). This derivative will, for. convenience, be referred to as the parnino salicylic acid (PAS) derivative; the said derivative is apparently not a true salt of the diazolone being coloured (whereas the diazolone and PAS themselves are colourless) and having a relatively low melting point viz. C. (dec.).

We shall now describe the physiological and like prop erties of our new'series of compounds.

Upon in Vitro tests 'on M. tuhercuiosis our new compounds in general show rather less inhibitory action than isonicotinic acid hydrazide itself. However upon in vivo tests carried out on man and upon animals the inhibitory effect of our new compounds is unexpectedly greater than that of isonicotinic acid hydrazide as judged by weight gain experiments, by pathological test on organs from animals treated by the new compounds and by X-ray examination of treated humans.

The toxicity of the new diazolone is also substantially less than that of isonicotinic' acid hydrazide both on oral administration and upon subcutaneous injection. Thus the 'LDso (subcutaneous) of the diazolone is approximately one tenth of that of isonicotinic acid hyd'ra'zzide when determined in the mouse and one third of that of isonicotinic acid hydrazide on oral administration in rabbits. The toxicity of the salts or salt-like derivatives is of course in part dependent upon whether the radical used to form the salts itself has any inherent toxicity.

The LDso of the diazolone upon subcutaneous injection into white mice has been determined to be .2650 mg./kg.

The diazolone and many of its non-toxic derivatives show no substantial side effects on administration to humans as is noticed with comparable dosages of for example streptomycin and p-amino salicylic acid.

One of the most important properties of our new conipounds is however the fact that they have been shown to have a very much lesser tendency to give rise to resistant strains of M. tuberculosis than substantially all of the tuberculostatics hitherto in common use. Arising from this property in particular we have obtained results in clinical trials upon the use of our new compounds by themselves which show striking improvement as against known tuberculostatics when the latter are used either singly, in admixture or consecutively.

The following is a description by way of example only of trials which we have carried out to demonstrate the last mentioned advantage of our new compound. The results of these trials will of course also illustrate the other advantages of the said new compounds.

In vitro tests were first carried out upon M. tubercu- 4 These results show that the PAS derivative of the diazolone is effective against these resistant strains at small dilutions at which isonicotinic acid hydrazide shows no effect.

losis (H.37 RV) to ascertain whether our new com- These in vitro experiments have been confirmed by pounds showed any tendency to produce resistant strains. animal experiments and clinical trials in several countries. Thus, the diazolone and its PAS derivative were tested, In these clinical trials it has been demonstrated that our by standard repeated subculturing techniques used for new compounds, particularly the diazolone itself and its this purpose (see for example Barnet et a1. Lancet, 1953, PAS derivative, are highly effective inter alia against I, 314, and Barry et al. Lancet, 1953, I, 978) in comparivery resistant strains of M. tuberculosis. A high proporson with isonicotinic acid hydrazide. Two separate extion of patients treated with the new compounds have periments were conducted with each tuberculostatic (tests shown improvement, in some cases markedly so, as a A and B) and the amount of each agent required to result of the treatment. This improvement has moreinhibit growth of each sub-culture was determined. The over occurred in cases which have failed to respond to results are shown in the following table (I): all hitherto known treatments including treatment with Table I Conan. of agent (1: g./ml.) required to inhibit growth of organism Sub-cultures Orig. cult. s

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 2-pyridyl-(4)-1,3,4-oxydiazo-{ A 0.1 0.1 0.1 0.1 0.1 1.0 0.1 0.1 1.0 1.0 1.0 1.0

mile-(5) B 0.1 0.1 0.1 0.1 1.0 1.0 1.0 1.0

These figures indicate that although in vitro the concentration of the new compounds required to inhibit growth of this particular strain of organism is with the initial culture as much as ten times greater than that of INH, with themunlike INHsubsequent cultures show almost no tendency to develop resistance, so much so that by the time the eleventh sub-culture is reached, while substantially the same amount of the diazolone or its PAS derivative will inhibit growth as is required to inhibit growth of the original culture, with isonicotinic acid hydrazide it is seen that the amount of this substance required after only ten sub-cultures to inhibit growth, is some 50,000 times that required to inhibit the original culture.

Tests were next carried out upon the in vitro action of the PAS derivative of our diazolone against two strains of M. tuberculosis known to have a strong resistance against isonicotinic acid hydrazide. In these tests the action of the PAS derivative is compared with the action of isonicotinic acid hydrazide (INH) at various dilutions. The results are shown in the following table (II):

Table II Strain Munehen Strain "Kornahl Dilution of tuberculostatie compound PAS salt of diazolone PAS INH INH "salt" Test 1 Test 2 1/10,000 1/20.000 M 3 l/l60.000 /320,000 l/640.000 ,2s0.000.. 5, A

(Control) In the above table: +++means strong growth. ++means moderate growth. +means weak growth. means no growth.

isonicotinic acid hydrazide, p-amino salicylic acid, streptomycin and terramycin. Thus for example, a group of 70 patients, including three moribund patients, who had already failed entirely to respond to high dosages of streptomycin, isonicotinic acid hydrazide, p-amino salicylic acid and terramycin showed in 70% of the cases marked improvement within 12 weeks of the commencement of treatment with the PAS derivative of 2-pyridyl-(4)-1,3,4- oxydiazolone-(S); the three moribund patients were able to leave their beds after a fortnight from commencement of treatment.

The dosage rate of our diazolone is conveniently from 800-1200 mg. per day for a 70 kg. man, and comparable doses of the derivatives may of course be used. The compounds are conveniently administered orally, for example in tablets or capsules or by inhalation or insufilation of aqueous solutions.

The new tuberculostatics according to the invention may be prepared by the reaction of phosgene with isonicotinic acid hydrazide to form the new diazolone which may then be transformed as desired into its salts or salt like derivatives.

According to the invention therefore there is provided a process for the preparation of new tuberculostatic compounds in which phosgene is reacted with isonicotinic acid hydrazide (INH) or a salt thereof.

The reaction preferably takes place in a suitable solvent for the INH or its salts, according to which is being used. In the case of INH salts the reaction is conveniently carried out in an aqueous medium, whilst in the case of INH itself a non-aqueous solvent such as dioxane may be used or dilute mineral acid is suitable. In the latter case water itself is also suitable as the reaction medium, since the introduction of phosgene into the aqueous medium will in fact give rise to the presence of hydrochloric acid.

The treatment with phosgene may be performed at any convenient temperature, for example between 0 C. and the boiling temperature of the mixture, preferably between room temperature and 50 C.

The equivalent amount or an excess of phosgene is introduced into the reaction mixture and the precipitated salt thus formed may then be isolated by filtration. It may be transformed in any convenient manner into the free compound e. g. by dissolving in aqueous alkali solutions and precipitation of the compound by acidification to a pH of about 7.

According to a further feature of the invention there is provided a process for the preparation of metalsalts or saltlike compounds of 2-pyridyl-(,4)-1,3,4-oxydiazOlone-(S) in which 2-pyridyl-(4)-1.3,4,-oxydiazolone- (5) or a salt thereof is reacted, preferably in aqueous solution, with a metal salt.

In many cases the resulting metal salt or salt-like compound is diflicultly-soluble and may readily be separated.

The preferred metal salts are those of copper, nickel, iron, cobalt, manganese and gold.

According to a still further feature of the invention there is provided a process for the preparation of the p-amino salicylic acid compound of 2-pyridyl-(4)-1,3,4- oxydiazolone-(S) in which the diazolone or a salt thereof is reacted with p-amino salicylic acid or a salt thereof.

It is preferred that an alkali metal, e. g. the sodium salt, of PAS and the hydrochloride of the 1,3,4-diazolone should be metathetically reacted in aqueous solution.

In order that the invention may be wellunderstood the following examples are now given by way of illustration only:

Example 1.P reparation of 2-pyridyl-(4)-1,3,4- oxydiazolone-(S) 5 gms. isonicotinic acid hydrazide are dissolved in 300 ml. of hot anhydrous dioxane. 80 gms. of gaseous phosgene are introduced at a temperature of 44 C. within 35 minutes. The new compound precipitates in the form of its hydrochloride and is filtered with suction. Yield 8.0 gms.

14 gms. of this salt are dissolved in 100 ml. aqueous caustic soda lye, treated with charcoal, filtered and then acidified with hydrochloric acid to pH 7. The precipitated substance is filtered with suction and dried. Yield 9 gins. It may be recrystallised from water or ethanol. Melting point 257 C. (with decomposition).

Analysis-3.664 mg. substance give 6.895 mg. CO2 and 0.960 mg. H2O. Calculated for C'zHsNaOz: C, 51.5%; H, 3.06%; N, 25.75%. Found: C, 51.35%; H, 2.95%; N, 25.63%.

Example 2.Preparati0n of 2-pyridyl-(4)-1,3,4-0xydiazolone-(S) 300 gms. isonicotinic acid hydrazide are dissolved in 600 ml. water and 170 ml. concentrated hydrochloric acid and the solution cooled to room temperature. The solution is then placed in a 2000 m1. flask equipped with an inlet tube of large diameter. In the course of 3 hours 1200 gms. of phosgene are fed into this solution while cooling and a precipitate is formed. The contents of the flask are then transferred into a 4000 ml. beaker, and the pH brought to 6 with 25% ammonium hydroxide while cooling. After cooling one hour at 5 C. the precipitate is filtered by suction, washed with water and dried. Yield: 340 gms. melting point 240 C. (with decomposition).

The product is purified by dissolving it in diluted ammonia and adding diluted nitric acid up to pH 6 when the product precipitates. It is also possible to recrystall-ise the product from water, alcohol or other suitable solvents. Melting point 265 C. (with decomposition). Example 3.-Preparation of 2-pyridyl-(4)-1,3,4-0xydiazolone-(S) using water as solvent 138 gms. isonicotinic acid hydrazide are dissolved in 1 1. water at room temperature, and phosgene is led in slowly with good stirring. The temperature rises and a white precipitate of the oxydiazolone is formed, which dissolved as the hydrochloride in the course of the reaction. The reaction is complete after an approximately equal weight (140 gms.) phosgene has been absorbed. On cooling the solution to 0 the hydrochloride crystallises out in a very satisfactory manner. The free base may be obtained by neutralising the solution of the hydrochloride with 50% potassium carbonate solution until it is slightly alkaline. The oxydiazolone'is precipitated as a'fine white precipitate and is filtered and Washed free of potassium salts with water. It may be crystallised from pyridine or alcohol, M. P. 274-276 (decomposition).

Example 4.-Preparati0n of para-aminosalicylic acid salt f 2 -P)" yl.- 4 -1 ,3 ,4-0xydiaz0l0n'e- (5) Example 5.Pr eparation of copper complex salt of 2-pyridyl-( 4 ,3,4-oxydiaz0lone- 5) 3 gms. 2-pyridyl-(4)-l,3,4-oxydiazolone-(5), obtained according to Example 1 or 2, are dissolved in ml. distilled water containing a trace of ammonia. 3 gms. CuSO'4.5H2O are dissolved in 100 ml. water and mixed with 5 ml. concentrated ammonium hydroxide. This solution'is added to the first solution at a temperature of '80-'-90 C. while stirring. A greenish blue precipitate is formed. The mixture is then cooled to room temperature, filtered with suction and the crystals thoroughly washed with boiling ethanol. Yield 3.2 gms., melting point 182 C. (with decomposition). The product is insoluble in alcohol, ether, acetone, dioxane; soluble in hot pyridine.

Example 6.Preparati0n of gold complex salt of v V 2 -pyridyl-(4)-1,3,4-Qxydiaz0l0ne-(5) 900 mg. of gold chloride are dissolved in 10 ml. 25 acetic acid. 2.0 gms. 2-pyridyl-(4)-1,3,4--oxydiazolone- (5) prepared as in Example 1 or 2, are dissolved in 30 ml. 1 N HCl with heating. The first solution is added to the second While stirring. A light yellow clear solution is obtained which upon standing one hour at -5 C. crystallises. Yield 900 mg; melting point 212-213 C. (with decomposition) Example 7.-Preparati0n of cobalt complex salt of Z-pyridyl- (4) -1,3,4-oxydiaz0l0ne-(5) 3.0 gms. CoCl2.6H2O and 5.0 gms. NH4C1 are dissolved in 20 m1. of distilled water. 2.0 gms. 2-pyridyl-(4)-1,3,4- oxydiazolone-(S) prepared as described in Examples 1 or 2 are dissolved in 20 ml. distilled water containing a trace of 25% ammonium hydroxide and added to the cobalt salt solution. An orange yellow precipitate is formed which after standing for several hours at 0 C. is filtered by suction. Yield 2.67 gms.; melting point above 350 C Example 8.Preparati0n of nickel complex salt of Z-pyridyl-(4)-1,3,4-0xydiaz0l0ne-(5) 4.00 gms. NiSO4.7HzO and 5.0 gms. NHiCl are dissolved in 20 ml. distilled water and admixed with 1 ml. 25% ammonium hydroxide. 2.00 gms. 2-pyridyl-(4)- 1,3,4-oxydiazolone-(5) are dispersed in 20 ml. distilled water and added with sufiicient 25% ammonium hydroxide to bring the whole in solution, filtered and admixed with the solution of the nickel salt. A light green precipitate is formed and isolated by filtration. Yield: 2.45 gms., melting point above 350 C Example 9.Preparation of manganese complex salt of Z-pyridyl- (4 ,3,4-0xydiazol0ne- (5 300 gms. MnSO4.7H2O and 5.0 gms. NH4C1 are dissolved in 20 ml. distilled water. 2.00 gms. 2-pyridyl-(4)- 1,3,4-oxydiazolone-(5) are dispersed in 20 ml. distilled water and added with 25 ammonium hydroxide solution in solution. The solution is filtered and added to the manganese salt solution. A cream coloured precipitate is formed immediately and filtered with suction. Yield: 2.6 ms; melting point above 350 C.

Example 10.-Preparation of iron complex salt of Z-pyridyl- (4 ,3,4-xydiaz0l0ne- Example 11.Preparation of copper salt complex of Z-pyridyl- (4) -1,3,4-0xydiaz0lone- (5) 2 gms. Z-pyridyl-(4)-1,3,4-oxydiazolone-(5) are suspended in 100 ml. distilled water and added with aqueous ammonium hydroxide until solution is complete. 3 gms. CuSOrfiI-IzO are dissolved in 100 ml. distilled water and added with ammonium hydroxide until a deep blue solution is formed. The copper solution is added to the oxydiazolone solution while stirring at 8090 C. Upon cooling, the product crystallises as beautiful greenish-blue crystals (plates). After standing 3 hours at room temperature the crystals are isolated by filtration with suction. Melting point 182 C. (with decomposition).

This application is a continuation-in-part of my application Serial No. 417,949 filed March 22, 1954, now abandoned.

I claim:

1. The p-amino salicyclic acid derivative of the compound Z-pyridyl-(4)-l,3,4-oxydiazo1one-(5), said derivative being orange colored and having a melting point of C. with decomposition.

2. A process for the production of a new antituberculotic compound of orange color and having a melting point of 165 C. with decomposition which comprises reacting a compound selected from the group consisting of para-amino salicyclic acid and salts thereof with a compound selected from the group consisting of 2-pyridyl- (4)-l,3,4-oxydiazolone-(5) and salts thereof and isolating said antituberculotic compound as the product of the reaction.

3. A process for the production of a new antituberculotic compound of orange color and having a melting point of 165 C. with decomposition which comprises reacting an alkali metal salt of para-amino salicyclic acid withan acid addition salt of 2-pyridy1-(4)-l,3,4-oxydiazolone-(5) and isolating said antituberculotic compound as the product of the reaction.

4. A' process as set forth in claim 3, wherein sodium p-amino salicylate is reacted with the hydrochloride of 2-pyridyl-(4)-1,3,4-oxydiazolone-(5) in an aqueous medium.

References Cited in the file of this patent UNITED STATES PATENTS 2,665,279 Aeschlimann et al Jan. 5, 1954 OTHER REFERENCES Panray: Amer. Rev. of Tuberculosis, vol. 67, p. 6, March 1953. 

1. THE P-AMINO SALICYCLIC ACID DERIVATIVE OF THE COMPOUND 2-PYRIDYL-(4)-1,3,4-OXYDIAZOLONE-(5), SAID DERIVATIVE BEING ORANGE COLORED AND HAVING A MELTING POINT OF 165* C. WITH DECOMPOSITION. 